SUMMARY Glioblastoma (GBM) tumors manifest with a large, proliferative core and a diffusely infiltrative border, the latter contributing to incomplete surgical resection and inevitable tumor recurrence. A better understanding of the specific molecular pathways responsible for cell infiltration in GBM may thus provide novel and potentially more effective opportunities for therapy. The biology of tumor cell infiltration/migration is complex and intimately linked to its microenvironment, including adaptations for unique metabolic, hypoxic, motility, extracellular matrix, and neuroinflammatory stressors. The pathways are difficult to recapitulate in glioma models that rely on cell culturing. As cell fate decisions converge on the level of epigenetic control, one attractive strategy to study the intricate biology of migration is to use epigenetics to define the transcriptional regulators of migration in a system that closely resembles the native tumor cell state. In this proposal, we take advantage of our lab's epigenetic expertise to study cell fate states in glioma stem cell populations (GSCs), directly isolated from their tumor niche without pre-culture artifact [37-38,57]. Through the unique comparison of open chromatin in freshly derived GSCs and normal germinal matrix progenitors, we were able to distinguish the transcriptionally accessible regions in GSCs that specifically relate to cell migration (in final revisions). This allowed us to infer the most salient transcription factor (TF) motifs within tumor-specific regulatory regions linked to migration, where the TEAD family emerged as a top candidate. The TEAD TFs, along with their co- activators YAP/TAZ, are the main effectors of the Hippo pathway, and have been studied for their oncogenic regulatory role predominantly outside of the brain. To test the functional role of YAP-TEAD in GBM growth and migration, we generated TEAD1 knockout in patient-derived GSCs using CRISPR/Cas9 and employed the YAP-TEAD inhibitor, Verteporfin, to inhibit TEAD activity. Using both methods to inhibit TEAD1 activity, we detected reduced capacity for cell migration in vitro and robust downregulation of EGFR and pERK expression. EGFR knockout similarly displayed reduced migration in vitro, as well as decreased TEAD activity. Based on this, here we hypothesize that TEAD activity regulates infiltrative growth in GBM, mediated at least partially through an EGFR regulatory loop. To test this hypothesis, we aim to 1) systematically define TEAD occupancy at open chromatin and downstream TEAD-target genes across GBM subtypes; 2a) dissect mechanistically the relationship between Hippo-YAP/TAZ-TEAD and EGFR/RTK-ERK; 2b) define the role of TEAD1 in regulating migration and growth in vitro and in/ex vivo, and its synergy with EGFR; and 3) test the potential therapeutic efficacy of YAP-TEAD inhibitors, such as Verteporfin. We envision YAP/TAZ-TEAD as an important regulator of infiltrative growth in GBM, where pharmacologic inhibition of its activity alleviates tumor burden while also blocking oncogenic RTK effects, thus offering new therapeutic options for patients with GBM.